1. Field of the Invention
This invention relates to a nucleic acid sequence encoding a functional module or domain of a particular peptidoglycan hydrolase, i.e., the φSH2 prophage endolysin, a protein which specifically attacks the cell wall peptidoglycan of untreated Staphylococcus aureus and coagulase negative staphylococci (S. chronogenes, S. epidermis, S. hyicus, S. simulans, S. warneri, and S. xylocus). The φSH2 prophage endolysin is active over a broad range of physiological conditions including the pH and calcium concentration of bovine milk. The invention further relates to methods of treating diseases caused by the bacteria for which the φSH2 prophage endolysin is specific.
2. Description of the Relevant Art
Staphylococcus is a genus of Gram-positive cocci that includes both human and animal pathogens. Multiple drug resistant strains of S. aureus (e.g., methicillin-resistant S. aureus, MRSA) pose a major threat to human health. A recent study reports clinical estimates indicating that MRSA strains caused more than 94,000 serious infections and more than 18,000 deaths in the United States in 2005 (Klevens et al. 2007. JAMA 298:1763-1771).
Also in the dairy industry, staphylococci are a persistent problem. Bovine mastitis, an infection of the mammary gland, results in annual losses between $1.7 billion and $2 billion in the United States alone (Sordillo et al. 2002. J. Mammary Gland Biol. Neoplasia 7 (2): 135-146). S. aureus is among the most relevant causative agents of this disease, accounting for 18% of mastitis cases in a study carried out on dairy herds in the states of New York and Pennsylvania (Wilson et al. 1997. J. Dairy Sci. 80: 2592-2598). The conventional method of treatment by antibiotics is often less than 50% effective and often leads to premature culling (Deluyker et al. 2005. J. Dairy Sci. 88:604-614). Furthermore, the use of broad range antibiotics such as pirlimycin and penicillin, which are commonly applied for treatment of mastitis (Cattell et al. 2001. J. Dairy Sci. 84:2036-2043), can contribute to the development of resistance in mastitis pathogens and nonrelated bacteria (Fischetti, V. A. 2005. Trends Microbiol. 13:491-496; Lee, J. H. 2003. Appl. Environ. Microbiol. 69:6489-6494; Vanderhaeghen et al. 2010. Epidemiol. Infect. 138:606-625). In an international study including several European countries and the United States, 57% of 811 S. aureus isolates from bovine mastitis were shown to be β-lactamase positive (De Oliveira et al. 2000. J. Dairy Sci. 83(4): 855-862). Moreover, there is a debate on whether antibiotic resistance can be transferred from farm animals to humans (Ferber, D. 2002. Science 295:27-28; Ferber, D. 2003. Science 301:1027).
In contrast to antibiotics, the use of pathogen-specific antimicrobials such as bacteriophage endolysins is expected to reduce the risk of resistance development (Walsh, C. 2003. Nat. Rev. Microbiol. 1:65-70). Bacteriophage endolysins are proteins which are produced inside an infected host cell at the end of the lytic multiplication cycle of the phage in order to lyse the bacterial cell from within, thereby releasing the phage progeny. In most cases, the lysis event is triggered by the action of a holin, another phage encoded protein which creates pores in the cytoplasmic membrane, enabling the endolysin to gain access to the host cell wall and degrade the peptidoglycan (Young and Blasi. 1995. FEMS Microbiol. Rev. 17:191-205). When exposed externally to Gram positive bacteria, in the absence of an outer membrane, these enzymes can also degrade the peptidoglycan, and lyse the cells. This makes them potential antimicrobials against Gram-positive pathogens such as Staphylococcus aureus (Fischetti, supra; Loessner et al. 2005. Curr. Opin. Microbiol. 8: 480-487). Furthermore, development of resistance in Gram-positive organisms against the highly specific action of phage endolysins is believed unlikely due to coevolution of phage and host, and up to now no resistant strains have been reported despite repeated efforts to find them (Fischetti, supra; Loeffler et al. 2001. Science 294:2170-2172; Schuch et al. 2002. Nature 418:884-889). Bacterial strains resistant against other (non-endolysin) peptidoglycan hydrolases such as the bacteriocin Lysostaphin (Dehart et al. 1995; Gründling et al. 2006. J. Bacteriol. 188:6286-6297; Sugai et al 1997. J. Bacteriol. 179: 4311-4318) or human lysozyme (Guariglia-Oropeza and Helmann. 2011. J. Bacteriol. 193:6223-6232; Vollmer, W. 2008. FEMS Microbiol. Rev. 32:287-306) have been described. Resistance against Lysostaphin has been ascribed in most cases to changes within the pentaglycine bridge (Dehart et al., supra; Rohrer et al. 1999. Proc. Natl. Acad. Sci. USA 96:9351-9356; Stranden et al. 1997. J. Bacteriol. 179:9-16; Sugai et al., supra; Thumm and Götz. 1997. Mol. Microbiol. 23:1251-1265), which is the target of Lysostaphin (Schindler and Schuhardt. 1964. Proc. Natl. Acad. Sci. USA 51:414-421) and presumably constitutes the most variable part of staphylococcal peptidoglycan (Schleifer and Kandler. 1972. Bacteriol. Rev. 36:407-477).
Endolysins from a Gram-positive background show a modular architecture, consisting of one or more enzymatically active domains, which cleave certain bonds within the bacterial peptidoglycan, and often a cell wall binding domain, which directs the enzyme to its substrate and confers specificity for the target cells. The latter is usually located at the C-terminus of the protein (Borysowski et al. 2006. Exp. Biol. Med. (Maywood) 231:366-377; Fischetti, supra; Loessner 2005, supra). According to the bonds cleaved by the enzymatically active domains, these domains can be classified into five different groups: (i) muramidases (also known as lysozymes) and (ii) glucosaminidases, which are both glycosidases and cleave the N-acetylmuramoyl-β-1,4-N-acetylglucosamine and N-acetylglucosaminyl-β-1,4-N-acetylmuramine bonds within the glycan strand of the peptidoglycan, respectively; (iii) lytic transglycosylases, which cleave the same bond as muramidases, but by a different mechanism; (iv) amidases, which cut between the glycan and the peptide moieties; and (v) endopeptidases, which cleave within the peptide moiety. The latter can be further divided into those enzymes cutting within the stem peptide, those cutting within the inter-peptide bridge, and those cleaving between the stem peptide and the inter-peptide bridge (Borysowski et al., supra; Hermoso et al. 2007. Curr. Opin. Microbiol. 10: 461-472; Loessner, M. J., supra). As demonstrated by several studies, the modular organization of phage endolysins allows artificial rearrangement of their functional domains, yielding protein chimeras with new and potentially optimized properties for control of pathogens (Becker et al. 2009b. Gene 443, 32-41; Croux et al. 1993a. Mol. Microbiol. 9:1019-1025; Croux et al. 1993b. FEBS Lett. 336:111-114; Diaz et al. 1990. Proc. Natl. Acad. Sci. U.S.A. 87:8125-8129; Donovan et al. 2006a. Appl. Environ. Microbiol. 72:2988-2996; Schmelcher et al. 2011. Microb. Biotechnol. 4:651-662). Therefore, bacteriophage endolysins do not only represent a promising alternative to antibiotics in their native form, but also a source of functional modules for the construction of tailor-made antimicrobials (Donovan et al. 2009. Biotech International 21:6-10).
The genomic sequence of Staphylococcus haemolyticus strain JCSC1435 was published recently, and two prophages were identified within the genome (Takeuchi et al. 2005. J. Bacteriol. 187:7292-7308). One of the prophages, φSH2, contains a gene (SH2333) coding for a putative endolysin, which was annotated as N-acetylmuramoyl-L-alanine amidase (BAE05642.1). Bioinformatic analysis suggests that this protein consists of two enzymatically active domains and one C-terminal cell wall binding domain. A conserved domain database (retrieved from the Internet: <URL: ncbi.nlm.nhi.gov) search identified a CHAP (Cysteine, Histidine-dependent Amidohydrolases/Peptidases) domain (pfam#PF05257; Bateman and Rawlings. 2003. EMBO J. 15:4789-4797; Rigden et al. 2003. Trends Biochem. Sci. 28:230-234) at the N-terminus, an Amidase—2 domain (N-acetylmuramoyl-L-alanine amidase; pfam#PF01510) in the center, and a bacterial SH3 domain (SH3b domain), which is associated with cell wall binding, at the C-terminus of the protein (pfam#PF08460; Whisstock and Lesk. 1999. Trends Biochem. Sci. 24:132-133). The crystal structure of one representative of the Amidase—2 family, the AmiE domain of the major S. epidermidis autolysin AtlE has recently been reported as the first protein structure with an amidase-like fold from a Gram-positive bacterial background, and also the structure of its binding domain and the phylogenetic relationship of the protein have been analyzed (Albrecht et al. 2012. J. Bacteriol. 194:2630-2636; Zoll et al. 2010. PLoS Pthog. 6: e1000807; Zoll et al. 2012. J. Bacteriol. 194:3789-3802). The φSH2 lysin shares its domain architecture with a number of other staphylococcal lysins described so far, such as the φ11 prophage endolysin LytA (Wang et al. 1991. Gene 102:105-109), the phage K endolysin LysK (O'Flaherty et al. 2005. J. Bacteriol. 187:7161-7164), the phage Twort endolysin plyTW (Loessner et al. 1998. FEMS Microbiol. Lett. 162:265-274), and the S. warneri phage WMY endolysin lysWMY (Yokoi et al. 2005. Gene 351:97-108). When more than fifty SH3b-containing staphylococcal peptidoglycan hydrolases (including the aforementioned phage endolysins) were classified based on overall sequence homology, the majority of the proteins fell into five groups featuring mostly >90% within-group but mostly <50% between-group sequence identity. The φSH2 lysin (previously referred to as “haemolyticus JCSC1435”) was one of six “stand-alone” proteins that shared less than 50% identity with any of the groups (Becker et al. 2009b, supra).
Peptidoglycan structure varies only slightly between different species and strains of the genus Staphylococcus, comprising a highly conserved stem peptide and a glycine-rich inter-peptide bridge (usually a penta- or hexa-glycine bridge) in which single residues can be replaced by L-serine or L-alanine (Schleifer and Kandler, supra). Therefore, it is not uncommon that an endolysin from a phage specific for a certain staphylococcal species also shows activity against other species of the genus, as demonstrated for lysWMY (Yokoi et al., supra) and LysK (Becker et al. 2009a. FEMS Microbiol. Lett. 294:52-60). S. haemolyticus peptidoglycan differs from that of S. aureus (pentaglycine bridge) by variations of the inter-peptide bridge, the most predominant cross bridges being COOH-Gly-Gly-Ser-Gly-Gly-NH2 and COOH-Ala-Gly-Ser-Gly-Gly-NH2 (Billot-Klein et al. 1996. J. Bacteriol. 178:4696-4703).
To counter the rise of drug resistant pathogenic bacteria, there is a need for new specific antimicrobial treatments. Reagents shown to be very specific for the genera, species or strains of concern would give better effective control of economically important diseases and therefore are ideal candidates for therapeutic treatments.